This invention relates generally to the field of pest management for agriculture. More particularly the invention relates to the use of certain proteases as pesticidal toxins, particularly for transgenic insecticidal protocols. The invention further comprises insecticidal compositions as well as transgenic techniques including novel expression constructs, vectors, methods for the infection of insects and delivery of toxins, and ultimately for the development of insect resistant transgenic plants.
One of the goals of agricultural research is to increase profitability of agriculture while decreasing its environmental impact. Integrated pest management programs need a diversity of control strategies and agents to maximize profits and minimize environmental damage. This need comes at a time when there has been a decrease in the diversity of classical pesticides. Although most experts agree that artificial pest control is needed to maintain our current level of agricultural productivity, over reliance on non selective pesticides has led to resistance, destruction of natural enemies, pest resurgence, and decreased profitability as well as environmental damage.
Synthetic chemical insecticides are effective for controlling pest insects in a wide variety of agricultural, urban, and public health situations. Unfortunately there are significant, often severe, side effects associated with the use of these products. Many pest populations have developed significant resistance to virtually all chemical insecticides, requiring higher and higher rates of usage for continued control. In a number of severe cases, highly resistant pest populations have developed which cannot be controlled by any available product. Chemical insecticides may also have deleterious effects on non-target organisms. Populations of beneficial arthropods, such as predators and parasites, are sometimes more severely affected by chemical applications than the pests themselves. Minor pests, ordinarily held in check by these beneficial organisms, may become serious pests when their natural constraints are removed by the use of chemical insecticides. Thus, new pest problems may be created by attempts to solve established problems.
Chemical insecticides may also have adverse effects on vertebrates. The use of DDT has been banned in the United States, due primarily to the insecticide""s great environmental persistence and its resulting tendency to accumulate in the tissues of predatory birds, thereby disrupting their ability to produce viable eggs. The use of carbofuran has been severely restricted due to its avian toxicity, and many species of fish are known to be quite sensitive to a variety of insecticides. A number of insecticides, such as methyl parathion, are also quite toxic to humans and other mammals, and by accident or misuse have caused a number of human poisonings. Clearly, the field of insect control would benefit greatly from the discovery of insecticides with improved selectivity for insects and reduced effects on non-target organisms.
The problems described above, along with other concerns including the possibility that some insecticides may act as human carcinogens, have created a strong demand for the development of safer methods of insect control.
Insect pathogens have been the objects of much study as potential pest control agents. Generally, these pathogens are quite selective for insects and in many cases affect only a few closely related species of insects. A number of insect pathogens have been developed as products, including bacteria (e.g., Bacillus thuringiensis and Bacillus popiliae), viruses (e.g., nucleopolyhedroviruses) and protozoa (e.g., the microsporidian Nosema locustae). These products occupy only a small fraction of the insecticide market. Although pathogens may ultimately cause a high level of mortality in pest populations, the insects may take weeks to die and continue to feed for much of that time. Thus, an unacceptably high level of crop or commodity damage may be inflicted before control is achieved. Currently, researchers are actively seeking ways to improve the effectiveness of insect pathogens and other biological control tools.
Insecticidal toxins from arthropods have been the objects of increasing interest over the past decade. These materials have proved useful for the detailed study of neural and neuromuscular physiology in insects. They have also been used to enhance the effectiveness of certain insect pathogens. The insecticidal toxin AaIT, from the scorpion Androctonus australis, has been employed for both purposes. This toxin belongs to a group of peptides that are lethal to a variety of insects but have no detectable effect in humans. Other toxins in A. australis venom are lethal to mammals but have no effect on insects. Understanding the molecular basis of this selectivity may lead to the development of chemical insecticides with reduced effects on mammals and other non-target organisms.
A number of transgenic protocols have been employed to help reduce the environmental impact of non-selective pesticides. A summary of current protocols follows.
One method involves transformation of plants with plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant variety can be transformed with a cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example Jones et al., Science 266: 789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).
The most extensively used heterologous gene for insect resistance involves the Bt endotoxin. A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence of a Bt Delta-endotoxin gene. Moreover, DNA molecules encoding Detla-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.
Other examples include use of a lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequences of several Clivia miniata mannose-binding lectin genes, use of a vitamin-binding protein, such as avidin. See PCT application US93/06487 the contents of which are hereby incorporated by reference. (The application teaches the use of avidin and avidin homologues as larvicides against insect pests); and use of an enzyme inhibitor, for example, a protease inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huub et al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci. Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus alpha-amylase inhibitor).
Still other recombinant strategies include use of insect-specific hormones or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344: 458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.
Further techniques include an insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor), and Pratt et al., Biochem. Biophys. Res. Comm. 163: 1243 (1989) (an allostatin is identified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encoding insect-specific, paralytic neurotoxins.
An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a chitinase, whether natural or synthetic has been used to create resistant transgenic plants. See PCT application WO 93/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al., Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.
A molecule that stimulates signal transduction is yet another class of proteins. For example, see the disclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess et al., Plant Physiol. 104: 1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone.
A hydrophobic mutant peptide has been used. See PCT application WO95/16776 (disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT application WO95/18855 (teaches synthetic antimicrobial peptides that confer disease resistance), the respective contents of which are hereby incorporated by reference.
A membrane permease, a channel former or a channel blocker has been used. For example, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993), of heterologous expression of a cecropin lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum. 
Yet another technique includes a viral-invasive protein or a complex toxin-derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. Rev. Phytopathol. 28: 451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.
An insect-specific antibody or an immunotoxin derived therefrom has been used. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. C f. Taylor et al., Abstract #497, Seventh Int""l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland, 1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).
Finally, a virus-specific antibody has been shown to confer protection. See, for example, Tavladoraki et al., Nature 366: 469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.
As can be seen from the foregoing there is a continuing need for environmentally safe alternatives to chemical pesticides.
It is an object of the present invention to provide genetically engineered insect pathogens which express a novel toxin.
It is yet another object of the invention to provide transgenic plants which express a non specific insect toxin to engineer insect resistant plants.
It is yet another object to provide expression constructs, vectors, and protocols for providing the insect pathogen and transgenic plants of the invention.
Other objects of the invention will become apparent from the detailed description of the invention which follows.
According to the invention, applicants have discovered that proteases which degrade or disrupt basement membranes such as metalloproteases, collagenases, gelatinases, stromelysins, cysteine proteases, as well as basement degrading proteases from snake venom, invertebrates, fungi, and bacteria are useful as insecticidal toxins. Surprisingly applicants have discovered that the basement membrane degrading proteases themselves act as toxins and may be used as insecticidal agents with efficacy against a variety of pest species.
When produced within insect tissues the protease is exported from the cells and degrades the basement membrane surrounding the tissues. Basement membranes provide structural support, a filtration function and a surface for cell attachment , migration and differentiation. Degradation of the basement membrane results in rapid death of the insect.
According to the invention polynucleotides are provided which include expression constructs for the expression of recombinant insecticidal proteases in insect pathogens or in transgenic plants. The expression constructs may comprise regulatory elements such as promoters and termination signals which are effective in the particular host cell or recipient (pathogen or plant) of the construct.
For purposes of this application the following terms shall have the definitions recited herein. Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5xe2x80x2 to 3xe2x80x2 orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.
As used herein the term xe2x80x9cbasement membrane degrading proteasexe2x80x9d shall include any protease capable of digesting or otherwise disrupting the basement membrane of a desired pest. This includes but is not limited to:
Mammalian matrix metalloproteases (MMPs) including:
Collagenases: (Interstitial collagenase (MMP-1, fibroblast collagenase, EC 3.4.24.7) Collagenase-3 (MMP-13) Neutrophil collagenase (MMP-5, EC 3.4.24.34) PMN-type collagenase (MMP-8);
Gelatinases(Gelatinase A (MMP-2, 72 kDa type IV collagenase, EC 3.4.24.24); Gelatinase B (MMP-9, 92 kDa type IV collagenase, EC 3.4.24.35))
Stromelysins: (Stromelysin-1 (MMP-3, transin, proteoglycanase, EC 3.4.24.17); Stromelysin-2 (MMP-10, transin-2, EC 3.4.24.22), Stromelysin-3 (MMP-11), Matrilysin (MMP-7, pump-1, EC 3.4.24.23); Metalloelastase (MMP-12); membrane-type MMP (MMP-14)
Mammalian cysteine proteases, including Cathepsin B (EC 3.4.22.1), Cathepsin L (EC 3.4.22.15), Cathepsin N
Snake venom proteases, including, Crotalus atrox (Western diamondback rattlesnake) and hemorrhagic metalloproteinases: (Ht-a, Ht-c, Ht-d, and Ht-e)
Invertebrate proteases, including: Hypoderma lineatum (fly) collagenase (EC 3.4.21.49), Uca pugilator (crab) collagenolytic endopeptidase (EC 3.4.21.32),
Fungal proteases, such as Entomophthora collagenase (EC 3.4.21.33)
Bacterial proteases, including:Clostridium histolyticum collagenase (EC 3.4.24.03), Streptomyces collagenase, Serratia marcescens cysteine endopeptidase. 
The term is also intended to include conservatively modified variants and other peptide variants which retain enzymatic activity of such proteases. The nucleotide sequences encoding these enzymes are generally known to those of skill in the art and available through sources such as Genbank. (see fibroblast collagenase, EC3.4.24.7 Genbank accession number X05231, PMN-type collagenase (MMP-8) Genbank accession number J05556, Gelatinase B (MMP-9, 92 kDa type IV collagenase, EC3.4.24.35 Genbank accession number J05070, Stromelysin-1 (MMP-3, transin, proteoglycanase, EC 3.4.24.17 Genbank accession number X05232), Stromelysin-2 (MMP-10, transin-2, EC 3.4.24.22, Genbank accession number X07820), Matrilysin (MMP-7, pump-1, EC 3.4.24.23 Genbank accession number X07819), Metalloelastase (MMP-12) Genbank accession number L23808, Cathepsin B (EC 3.4.22.1) Genbank accession number M14221, Cathepsin L (EC 3.4.22.15) Genbank accession number L23808, Crotalus atrox (Western diamondback rattlesnake) hemorrhagic metalloproteinases: Ht-a, Ht-c, Ht-d, and Ht-e Accession numbers:Ht-a: U01234, Ht-c: U01236, Ht-d: U01237, Uca pugilator (crab) collagenolytic endopeptidase (EC 3.4.21.32) Genbank accession number U49931 (accession # for the Uca enzyme), and Clostridium histolyticum collagenase (EC 3.4.24.03) Genbank accession number D29981. Those of skill in the art will appreciate that other basement membrane degrading proteases will be applicable to the teachings herein, or will become available or isolated using no more than routine experimentation.
By xe2x80x9camplifiedxe2x80x9d is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Canteen, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
The term xe2x80x9cconservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variationsxe2x80x9d and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine; and UGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and is within the scope of the present invention.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for its native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins W. H. Freeman and Company.
By xe2x80x9cencodingxe2x80x9d or xe2x80x9cencodedxe2x80x9d, with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the xe2x80x9cuniversalxe2x80x9d genetic code. However, variants of the universal code, such as are present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum, or the ciliate Macronucleus, may be used when the nucleic acid is expressed therein.
When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)). Thus, the maize preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants are listed in Table 4 of Murray et al., supra.
As used herein, xe2x80x9cheterologousxe2x80x9d in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
By xe2x80x9chost cellxe2x80x9d is meant a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells or insect cells.
The term xe2x80x9chybridization complexxe2x80x9d includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
The term xe2x80x9cintroducedxe2x80x9d in the context of inserting a nucleic acid into a cell, means xe2x80x9ctransfectionxe2x80x9d or xe2x80x9ctransformationxe2x80x9d or xe2x80x9ctransductionxe2x80x9d and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
The term xe2x80x9cisolatedxe2x80x9d refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment. The alteration to yield the synthetic material can be performed on the material within or removed from its natural state. For example, a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by means of human intervention performed within the cell from which it originates. See, e.g., Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868. Likewise, a naturally occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced by non-naturally occurring means to a locus of the genome not native to that nucleic acid. Nucleic acids which are xe2x80x9cisolatedxe2x80x9d as defined herein, are also referred to as xe2x80x9cheterologousxe2x80x9d nucleic acids.
As used herein, xe2x80x9cnucleic acidxe2x80x9d or xe2x80x9cnucleotidexe2x80x9d includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
As used herein xe2x80x9coperably linkedxe2x80x9d includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
As used herein, the term xe2x80x9cplantxe2x80x9d can include reference to whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells, seeds and progeny of same. Plant cell, as used herein, further includes, without limitation, cells obtained from or found in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Particularly preferred plants are agricultural plants.
As used herein, xe2x80x9cpolynucleotidexe2x80x9d includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons as xe2x80x9cpolynucleotidesxe2x80x9d as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides are not entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well.
As used herein xe2x80x9cpromoterxe2x80x9d includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9cplant promoterxe2x80x9d is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as xe2x80x9ctissue preferredxe2x80x9d. Promoters which initiate transcription only in certain tissue are referred to as xe2x80x9ctissue specificxe2x80x9d. A xe2x80x9ccell typexe2x80x9d specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An xe2x80x9cinduciblexe2x80x9d or xe2x80x9crepressiblexe2x80x9d promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of xe2x80x9cnon-constitutivexe2x80x9d promoters. A xe2x80x9cconstitutivexe2x80x9d promoter is a promoter which is active under most environmental conditions.
As used herein xe2x80x9crecombinantxe2x80x9d includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term xe2x80x9crecombinantxe2x80x9d as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
As used herein, a xe2x80x9cexpression cassettexe2x80x9d is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.
The term xe2x80x9cresiduexe2x80x9d or xe2x80x9camino acid residuexe2x80x9d or xe2x80x9camino acidxe2x80x9d are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively xe2x80x9cproteinxe2x80x9d). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
As used herein, xe2x80x9ctransgenic plantxe2x80x9d includes reference to a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. xe2x80x9cTransgenicxe2x80x9d is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term xe2x80x9ctransgenicxe2x80x9d as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
As used herein, xe2x80x9cvectorxe2x80x9d includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
A xe2x80x9cstructural genexe2x80x9d is a DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
The term xe2x80x9cexpressionxe2x80x9d refers to biosynthesis of a gene product. Structural gene expression involves transcription of the structural gene into mRNA and then translation of the mRNA into one or more polypeptides.
A xe2x80x9ccloning vectorxe2x80x9d is a DNA molecule such as a plasmid, cosmid, or bacterial phage that has the capability of replicating autonomously in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
An xe2x80x9cexpression vectorxe2x80x9d is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements including promoters, tissue specific regulatory elements, and enhancers. Such a gene is said to be xe2x80x9coperably linked toxe2x80x9d the regulatory elements.
A xe2x80x9crecombinant hostxe2x80x9d may be any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned genes in the chromosome or genome of the host cell.
A xe2x80x9ctransgenic plantxe2x80x9d is a plant having one or more plant cells that contain an expression vector. Plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture such as single cells, protoplasm, embryos, and callus tissue. The plant tissue may be in plant or in organ, tissue, or cell culture. These proteins can be used in techniques described herein as molecular markers in breeding to identify and/or select plants with improved insect resistance.
As used herein the term xe2x80x9csubstantially resistantxe2x80x9d refers to the fact that the transformed and transgenic plants of this invention have resistance to pests that invade, infect, or consume the particular plant species when compared to the corresponding non-transgenic or non-transformed plant.